Enhancing polyethylenimine's delivery of plasmid DNA into mammalian cells

Abstract

The effect of various chemical modifications of nitrogen atoms on the efficiency of polyethylenimines (PEIs) as synthetic vectors for the delivery of plasmid DNA into monkey kidney cells in vitro has been systematically investigated. The resultant structureactivity relationship has both provided mechanistic insights and led to PEI derivatives with markedly enhanced performance. For example, N-acylation of PEI with the molecular mass of 25 kDa (PEI25, one of the most potent polycationic gene delivery vectors) with alanine nearly doubles its transfection efficiency in the presence of serum and also lowers its toxicity. Furthermore, dodecylation of primary amino groups of 2-kDa PEI yields a nontoxic polycation whose transfection efficiency in the presence of serum is 400 times higher than the parent's and which exceeds 5-fold even that of PEI25.I nterest in gene therapy has soared in recent years because of its great promise in treating diseases ranging from inherited disorders to acquired conditions and cancer (1-3). However, much progress remains to be made before safe and reliable gene delivery vehicles are developed (4). Side reactions provoked by the viral elements, e.g., immune response and insertional mutagenesis (5), have prompted efforts to improve nonviral delivery systems (vectors) (6-8). Synthetic vectors based on polycations are particularly attractive because of their potential safety, nucleic acid cargo capacity, and designability (9-11). And yet, currently available polycations are severely limited by their low efficiencies (4,8).Improvements in gene delivery by polyplexes (polycation͞ DNA complexes) may come from a detailed examination of their mechanism of action, establishing the underlying structureactivity relationships, and manipulation of the vectors accordingly (12-16). Polyethylenimine (PEI), a readily available synthetic polycation introduced for transfection a few years ago (17), is an ideal candidate for such mechanistic investigations owing to its already relatively high transfection efficiency and ease of functionalization.Elucidation of the mechanism of PEI-mediated gene delivery has received much attention in recent years (18-24). Since every third atom in PEI is a protonatable nitrogen, the overall protonation level should rise when the pH drops to Ϸ5 inside the lysosomal compartment, leading to the influx of chloride ions and, consequently, to osmotic swelling and rupture of the lysosome. The early escape of the PEI͞DNA complex from the lysosome, and thus the avoidance of lysosomal degradation arising from the ''proton sponge effect,'' was postulated to be the cause of the high transfection efficiency exhibited by PEIs (17); this concept, however, has been recently challenged (22).In the present study, we carried out systematic chemical modifications of commercial branched PEIs with the goal of understanding and enhancing PEI-mediated transfection. PEIs with molecular masses of 25 and 2 kDa were selected because they exhibit high and low transfection efficiencies, respectively (17)...

Simple Modifications of Branched PEI Lead to Highly Efficient siRNA Carriers with Low Toxicity

“…Interestingly, partial acetylation of PEI amino groups was shown to enhance gene transfer activity (16). N-Acylation of PEI lowered its toxicity (17). Also, conjugation of acetate, butanoate, and hexanoate to branched PEI at low degree of substitution (below 25%) resulted in moderate improvement of transfection activity as demonstrated by Putnam and colleagues (18).…”

Modified polyethyleneimine with histidine–lysine short peptides as gene carrier

“…8 For example, it has been shown that PEI 25 kDa can efficiently lyse endosomes and facilitate endosomal escape of vector/ pDNA complexes. 9 However, in low-molecular-weight PEIs (for example, 10 kDa), the endosomolytic effect is not strong enough to release all PEI/DNA complexes. 8,10 Despite this inefficiency, PEI 10 kDa is a much safer polymer in comparison with PEI 25 kDa because of its significantly lower cell toxicity.…”

Quantitative comparison of polyethylenimine formulations and adenoviral vectors in terms of intracellular gene delivery processes

“…[3][4][5][6][7][8][9][10][11] A branched polymer containing primary, secondary, and tertiary amines, PEI is capable of complexing plasmid DNA and delivering it intracellularly to cells in vitro. The amine groups of PEI are thought to act as proton sponges, preventing endosomal acidification, which results in osmotic swelling and disruption of endosomal membranes.…”

“…Dodecylation, the addition of a 12-carbon alkyl chain onto PEI, is hypothesized to increase cytoplasmic delivery by raising cellular uptake of PEI by making the polymer more hydrophobic. 9 Recent data in COS-7 cells showed that dodecylation significantly increased cellular uptake of complexes over unmodified PEI, 9 but the intracellular steps responsible for the improvement have not yet been directly ascertained. One aim of our new work here, therefore, is to determine which of the intracellular delivery pathway parameters were most usefully enhanced by this chemical modification strategy.…”